Amit Pratush » Публикация
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Опубликовано
2013-10-14
Опубликовано на SciPeople2013-10-14 14:44:30
ЖурналAppl Biochem Biotechnol
Cloning, sequencing and expression of NHase gene of mutant strain of Rhodococcus rhodochrous PA-34 in E.coli
Аннотация
The NHase encoding gene of mutant 4D was isolated by PCR amplification. The
NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by
using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the
co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed
with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high
homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli
cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw)
whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria–
Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The
recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified
with a yield of 56 % with specific activity of 37.1 U/mg protein.